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In Message Tue, 26 Oct 1993 17:54:39 -0400,
Kristi R Harkins <kharkins@IASTATE.EDU> writes:
>
> We would like to use our conventional 752 with MDADS I to sort
>chromosomes. In the manual the company notes an analysis rate of 10,000/sec
>and sort rates of 5,000/sec. We have not yet had the opportunity to push
>the system to see if these numbers are real. Could anyone give me some real
>case scenario's or point me to a reference where I can find out actual sort
>rates, sample flow rates, 1, 2, or 3 droplet sorting, sheath PSI, crystal
>frequency, final purity, and what original percent of the population was
>being sorted???
When we sorted tomato chromosomes "Preparation and flow cytometric analysis
of metaphase chromosomes of tomato" Arumuganathan, et. al. Theor Appl Genet
(1991) 82:101-111 we used analysis rates of not more than 300/sec, sort
rates ~10-150/sec depending on the population to be sorted,
1 droplet sorting, 13 psi, 76micron quartz tip, 32kHz, and
coincidence detect on. Only ~2% of the original population was sorted and
purity was high. "High purity" is hard to evaluate as there is not a high
enough yield to be able to reanalyze. Some of the samples went to subsequent
PCR reactions and based on those results as well as chromosomes
sorted onto filter paper having very uniform distributions we were confident
that purity was 98% +. We were willing to work at slow rates for high
purity. Although we considered doing tandem sorts we never felt the original
yields on the chromosomes (absolute quantity) justified this. Because the
karyotype is not very well defined (both the size of the chromosomes and
AT/GC ratios in the tomato are fairly uniform) we had to analyze slowly to
retain CVs of ~2%. CV's of beads (Coulter DNA Check) were <1% before
analyzing chromosomes. Chromosome concentrations were also a problem for us
and were also part of the reason for slow sorts.
At Scott Cram and Joe Fawcett's SAC 1990 tutorial they give the
following rates as standard for human chromosomes:
Analysis 1250/sec
Homolog 52/sec
coincidence 5/sec
sort rate 47/sec
Chromosome # conc 2.4E7/ml
homolog conc 1.0E6/ml
>
> Along these same lines, if it were possible to tag a specific chromosome
>within a mixture, do you have to use a counter stain to see all the other
>unwanted chromosomes? I am concerned about the DNA fluorescence crossing over
>into the tag fluorescence channel. My reasoning stems from the idea that you
>could calculate the rate of coincidence based upon the ratio of the
>desired chromosome to the undesired ones and adjust the flow rate to minimize
>coincidence. Also, if you set the cytometer up with a bead which correlates
>in fluorescence intensity with what you estimate as the fluorescence
>equivalent to your tagged chromosome to maximize signal to noise, is this
>one signal enough to sort by??? I realize these questions are somewhat
>abstract, but if anyone has some experience with low-level fluorescence
>detection and sorting combined, I would appreciate hearing about your
>experiences.
>
>Thanks,
>---
I believe you would still have to counterstain. I suppose some others are better
suited to answer this but I think it is faulty to assume an evenly
distributed occurrence of coincidence. That being the case it is difficult
to say if anything else is coming along with your bead sorted population.
That would of course be easy to check if you do the sort.
What I have seen with much in situ work and single molecule labelling
with microscopy is that non-specific binding is a huge, not fully addressed
problem. That being the case having a second measure of the chromosome is
going to be of practical importance.
Hope this helps and that I have not babbled too much. If you have any
questions please feel free to call or e-mail.
**********************************************************************
James P. Slattery Tel 607-254-4862
Director, Flow Cytometry and Imaging Fax 607-255-2428
Director, Computing Facility Internet: jps4@cornell.edu or
Cornell Biotechnology CAT jim_slattery@qmrelay.mail.cornell.edu
Cornell University
Biotechnology Building
Ithaca, NY 14853 USA
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